670. Mutation Independent Silencing of Genes Involved in Retinal Degeneration by RNA Interference (RNAi) and Trans Complementation by a Codon Exchanged RNAi-Resistant Transgene

Abstract

More than one hundred different mutations in the gene encoding rhodopsin are associated with a group of retinal degenerations including retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Given this large heterogeneity of mutations, it would be ideal to develop mutation independent therapies for these diseases. We describe use of the endogenous RNA interference (RNAi) machinery present in eukaryotic cells to silence mutant and wild type rhodopsin alleles by a vector expressing a short hairpin RNA (shRNA). We found that in the human embryonic retinoblast cell line 911, wild type human rhodopsin driven by a CMV promoter could be silenced by 94.34|[plusmn]|2.17% and the most common rhodopsin-associated mutation in the United States, P23H, could be silenced by 94.9|[plusmn]|1.9% by a shRNA targeting rhodopsin. When the degeneracy of the genetic code was used to exchange 10 of 21 nucleotides of the region that encodes for the shRNA target in human rhodopsin cDNA, we found that the shRNA was no longer able to silence the codon exchanged rhodopsin mRNA (cmRNA). Moreover, the efficiency of translation of this cmRNA was equivalent to wild type rhodopsin mRNA.